Isolation and molecular characterization of mycotoxigenic fungi in agarwood

Agarwood (Oudh), is often used by people in the Kingdom of Saudi Arabia. The Oudh has been mentioned in the Hadith and is traditionally used for its aroma (perfuming smell) and potential medicinal applications. The aim of the study was to isolate mycotoxigenic fungi that grow on agarwood and the factors and storage conditions that enhance their growth potential. In addition to the detection of associated mycotoxins like: Aflatoxin B1 (AFB₁) and ochratoxin A (OTA) from agarwood. Agarwood samples were collected from local markets of Jeddah governorate, Kingdom of Saudi Arabia. Standard dilution plate method was used for the isolation of fungi. Isolated fungi were identified based on morphological characteristics and confirmed using molecular biology techniques. AFB₁ and OTA were detected by High Performance Liquid Chromatography (HLPC). The results indicated that the most commonly isolated fungal genera were in the following descending order: Aspergillus, Penicillium, Fusarium and Rhizopus. Among Aspergillus genera, A. flavus and A. ochraceus were detected based on their morphology and confirmed by PCR using specific primers. It was also noted that AFB₁ was released by 15.3 and 55.0% of A. flavus and A. parasiticus isolates respectively with levels reaching up to 14.60 µg/L. The moisture content in the samples ranged from 3% to 10% affected fungal growth. AFB₁ was detected in 22 out of 50 of the samples. The maximum level of AFB₁ (50.7 µg/kg) was detected in samples with higher moisture content (12%) stored at a temperature of 32 °C. Aspergillus fungi were found to be the most predominant fungal genera found on agarwood. Moisture content (9–10%) and storage temperature (32 °C) stimulated fungal growth and their ability to produce mycotoxins. For this reason, storage conditions at the marketing place should be adequate in order not to provide a conducive environment for fungal growth which is associated with the mycotoxin production. In order to prevent fungal growth and mycotoxin production, it would be recommended to store agarwood at temperatures not exceeding 25 °C and moisture content of up to a maximum of 5–6%.     

Mitigating N2O emissions from agricultural soils with fungivorous mites

Nitrous oxide (N₂O) is an important greenhouse gas and an ozone-depleting substance. Due to the long persistence of N₂O in the atmosphere, the mitigation of anthropogenic N₂O emissions, which are mainly derived from microbial N₂O-producing processes, including nitrification and denitrification by bacteria, archaea, and fungi, in agricultural soils, is urgently necessary. Members of mesofauna affect microbial processes by consuming microbial biomass in soil. However, how microbial consumption affects N₂O emissions is largely unknown. Here, we report the significant role of fungivorous mites, the major mesofaunal group in agricultural soils, in regulating N₂O production by fungi, and the results can be applied to the mitigation of N₂O emissions. We found that the application of coconut husks, which is the low-value part of coconut and is commonly employed as a soil conditioner in agriculture, to soil can supply a favorable habitat for fungivorous mites due to its porous structure and thereby increase the mite abundance in agricultural fields. Because mites rapidly consume fungal N₂O producers in soil, the increase in mite abundance substantially decreases the N₂O emissions from soil. Our findings might provide new insight into the mechanisms of soil N₂O emissions and broaden the options for the mitigation of N₂O emissions.Subject terms: Climate-change ecology, Climate-change ecology     

Fungal Diversity in Deep Sea Sediments from East Yap Trench and Their Denitrification Potential

Abstract

In recent years, the hadal trenches have been recognized as biological hot spots for deep sea researchers. Due to high hydrostatic pressure, low temperatures, high salinity and low nutrients, the microorganisms in hadal trenches may have unique community structure with potential for biotechnical application. Compared with bacteria and archaea, the diversity and ecological roles of fungi in hadal trenches remain largely unknown. The purpose of this study was to explore fungal diversity in deep-sea sediments of the Yap trench and their denitrification potential. In the present study, a total of 106 fungal strains were isolated from six sediment samples collected in the East Yap Trench. These fungi belonged to five classes (Dothideomycetes, Eurotiomycetes, Sordariomycetes, Cystobasidiomycetes, and Microbotryomycetes), thirteen genera (Acremonium, Alternaria, Aureobasidium, Aspergillus, Cladosporium, Cystobasidium, Engyodontium, Gliomastix, Lecanicillium, Penicillium, Phoma, Rhodotorula and Trichoderma) and eighteen species, based on morphological identification and ITS-rDNA sequence analysis. Among them, the dominant genus is Cladosporium, which accounting for 42.45% of the total fungal strains. Meanwhile, the denitrification potential of the fungal strains was also examined with two different denitrifying media (nitrate and nitrite as sole substrate, respectively). Two fungal strains (Acremonium sp. and Aspergillus versicolor), were found to be able to produce N₂O ex situ in the presence of nitrite. No fungus was found to produce N₂O by using nitrate. Our results suggest that fungi in hadal sediments, play important roles in nitrogen cycles.     

Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

Diverse Deep-Sea Fungi from the South China Sea and Their Antimicrobial Activity

We investigated the diversity of fungal communities in nine different deep-sea sediment samples of the South China Sea by culture-dependent methods followed by analysis of fungal internal transcribed spacer (ITS) sequences. Although 14 out of 27 identified species were reported in a previous study, 13 species were isolated from sediments of deep-sea environments for the first report. Moreover, these ITS sequences of six isolates shared 84–92 % similarity with their closest matches in GenBank, which suggested that they might be novel phylotypes of genera Ajellomyces, Podosordaria, Torula, and Xylaria. The antimicrobial activities of these fungal isolates were explored using a double-layer technique. A relatively high proportion (56 %) of fungal isolates exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among four marine pathogenic microbes (Micrococcus luteus, Pseudoaltermonas piscida, Aspergerillus versicolor, and A. sydowii). Out of these antimicrobial fungi, the genera Arthrinium, Aspergillus, and Penicillium exhibited antibacterial and antifungal activities, while genus Aureobasidium displayed only antibacterial activity, and genera Acremonium, Cladosporium, Geomyces, and Phaeosphaeriopsis displayed only antifungal activity. To our knowledge, this is the first report to investigate the diversity and antimicrobial activity of culturable deep-sea-derived fungi in the South China Sea. These results suggest that diverse deep-sea fungi from the South China Sea are a potential source for antibiotics’ discovery and further increase the pool of fungi available for natural bioactive product screening.     

Nitrous oxide in brackish Lake Nakaumi, Japan II: the role of nitrification and denitrification in N2O accumulation

In order to understand the role of nitrification and denitrification in the accumulation of nitrous oxide (N₂O) in the hypolimnetic water of brackish Lake Nakaumi, the effects of dissolved oxygen (DO) concentration on these activities were investigated by incubation experiments. N₂O was produced during the oxidation of NH₄ ⁺ to NO₂ ⁻ in nitrification and during the reduction of NO₃ ⁻ to N₂ in denitrification. N₂O-producing activity by nitrification (N₂O_N) increased markedly with decreasing concentrations of DO. Low DO (10%–30% saturation) induced high N₂O_N. In contrast to nitrification, N₂O-producing activity by denitrification (N₂O_D) decreased with decreasing concentrations of DO. Little N₂O was accumulated during denitrification under low-level conditions of DO (10%–30%), because of further reduction of N₂O to N₂. It can therefore be assumed that N₂O produced as the by-product of nitrification is concurrently reduced to N₂ by denitrification under low-DO conditions. This would result in no substantial accumulation of N₂O during active nitrification in the hypolimnetic water of Lake Nakaumi. Received: July 6, 2001 / Accepted: December 10, 2001     

Microbial Diversity of Some Sabkha and Desert Sites in Saudi Arabia

Several studies isolated fungal and bacterial species from extreme environments, such as Sabkha and hot deserts, as their natural habitat, some of which are of medicinal importance. Current research aimed investigating the microbial (fungi and bacteria) diversity and abundance in Sabkha and desert areas in Saudi Arabia. Soil samples from nine different geographical areas (Al-Aushazia lake, AlQasab, AlKasar, Tabuk, Al-Kharj, Al-Madina, Jubail, Taif and Abqaiq) were collected and cultured for microbial isolation. Isolated fungi and bacteria were identified by molecular techniques (PCR and sequencing). Based on 18S rDNA sequencing, 203 fungal species belonging to 33 genera were identified. The most common fungal genera were Fusarium, Alternaria, Chaetomium, Aspergillus Cochliobolus and Pencillium, while the most common species were Chaetomium globosum and Fusarium oxysporum. By 16S rDNA sequencing 22 bacterial species belonging to only two genera, Bacillus and Lactobacillus, were identified. The most commonly isolated bacterial species were Bacillus subtilis and Lactobacillus murinus. Some fungal species were confined to specific locations, such as Actinomyces elegans, Fusarium proliferatum, Gymnoascus reesii and Myzostoma spp. that were only isolated from Al-Aushazia soil. AlQasab soil had the highest microbial diversity among other areas with abundances of 23.5% and 4.4% of total fungi, and bacteria, respectively. Findings of this study show a higher degree of fungal diversity than that of bacteria in all studied areas. Further studies needed to investigate the connection between some isolated species and their habitat ecology, as well as to identify those of medicinal importance.     

Nitrification inhibitors effectively target N2O-producing Nitrosospira spp. in tropical soil

The nitrification inhibitors (NIs) 3,4-dimethylpyrazole (DMPP) and dicyandiamide (DCD) can effectively reduce N₂O emissions; however, which species are targeted and the effect of these NIs on the microbial nitrifier community is still unclear. Here, we identified the ammonia oxidizing bacteria (AOB) species linked to N₂O emissions and evaluated the effects of urea and urea with DCD and DMPP on the nitrifying community in a 258 day field experiment under sugarcane. Using an amoA AOB amplicon sequencing approach and mining a previous dataset of 16S rRNA sequences, we characterized the most likely N₂O-producing AOB as a Nitrosospira spp. and identified Nitrosospira (AOB), Nitrososphaera (archaeal ammonia oxidizer) and Nitrospira (nitrite-oxidizer) as the most abundant, present nitrifiers. The fertilizer treatments had no effect on the alpha and beta diversities of the AOB communities. Interestingly, we found three clusters of co-varying variables with nitrifier operational taxonomic units (OTUs): the N₂O-producing AOB Nitrosospira with N₂O, NO₃⁻, NH₄⁺, water-filled pore space (WFPS) and pH; AOA Nitrososphaera with NO₃⁻, NH₄⁺ and pH; and AOA Nitrososphaera and NOB Nitrospira with NH₄⁺, which suggests different drivers. These results support the co-occurrence of non-N₂O-producing Nitrososphaera and Nitrospira in the unfertilized soils and the promotion of N₂O-producing Nitrosospira under urea fertilization. Further, we suggest that DMPP is a more effective NI than DCD in tropical soil under sugarcane.     

Soil fungal diversity in three nature reserves of Jiuzhaigou County,Sichuan Province,China

Jiuzhaigou County is located at the southern transition zone of Sichuan Basin and the Qinghai–Tibet Plateau and is the site of three famous nature reserves, namely, the Jiuzhaigou Nature Reserve (JZNR), Baihe Nature Reserve (BHNR) and Wujiao Nature Reserve (WJNR). The soil fungal diversity in this region has not yet been investigated. In this study, we collected 25 soil samples from these three nature reserves. Soil fungi were isolated using the soil dilution plate technique and Rose Bengal agar medium. The culturable soil fungal density based on analysis of the 25 samples ranged from 2.18 log to 4.38 log CFU g^?1 dry weight soil, with the fungal density being highest in samples from JZNR and lowest in those from BHNR. Based on morphological characters and the results of phylogenetic analysis of the internal transcribed spacer (ITS) of the rDNA operon, we identified 38 genera (two genera could not be identified) belonging to Ascomycota, Zygomycota and Basidiomycota. The dominant genera were Penicillium, Humicola, Aspergillus and Trichoderma. The species richness index S, biodiversity index H′ and evenness index E of the 25 sampling sites were in the range 10–29, 1.96–3.05 and 0.74–0.95, respectively. The highest mean values of the S, H′ and E indices were in soil samples from BHNR, where the values of these indices were 20.00, 2.66 and 0.90, respectively. These results indicate that the diversity of culturable fungi in these three nature reserves was high. Furthermore, a total 14 Trichoderma isolates were tested for their antagonism activity against mycelium growth of three pathogens: Bipolaris maydis, Curvularia lunata, Rhizoctonia solani. The results showed that six Trichoderma isolates had good antagonistic effects on the three pathogenic fungi.     

Novel and highly diverse fungal endophytes in soybean revealed by the consortium of two different techniques

Fungal endophytes were isolated from the leaves of soybean cultivars in Brazil using two different isolation techniques — fragment plating and the innovative dilution-to-extinction culturing — to increase the species richness, frequency of isolates and diversity. A total of 241 morphospecies were obtained corresponding to 62 taxa that were identified by analysis of the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA). The Phylum Ascomycota predominated, representing 99% and 95.2% of isolates in the Monsoy and Conquista cultivars, respectively, whereas the Phylum Basidiomycota represented 1% and 4.8% of isolates, respectively. The genera Ampelomyces, Annulohypoxylon, Guignardia, Leptospora, Magnaporthe, Ophiognomonia, Paraconiothyrium, Phaeosphaeriopsis, Rhodotorula, Sporobolomyces, and Xylaria for the first time were isolated from soybean; this suggests that soybean harbours novel and highly diverse fungi. The yeasts genera Rhodotorula and Sporobolomyces (subphylum Pucciniomycotina) represent the Phylum Basidiomycota. The species richness was greater when both isolation techniques were used. The diversity of fungal endophytes was similar in both cultivars when the same isolation technique was used except for Hill’s index, N₁. The use of ITS region sequences allowed the isolates to be grouped according to Order, Class and Phylum. Ampelomyces, Chaetomium, and Phoma glomerata are endophytic species that may play potential roles in the biological control of soybean pathogens. This study is one of the first to apply extinction-culturing to isolate fungal endophytes in plant leaves, thus contributing to the development and improvement of this technique for future studies.     

Aerobiological investigations inside repositories of the National Archive of the Republic of Cuba

The objectives of this study were to evaluate the microbial prevalence inside six repositories of the National Archive of the Republic of Cuba in 2?months of the year and to examine some of the physiological features of fungi isolated in order to evaluate their potential for biodeterioration. The microbiological sampling was conducted in February and September using a slit impactor as air sampler. Appropriate selective culture media were used to isolate fungi and bacteria. Temperature and relative humidity were measured during the samplings. The cellulolytic activity and the production of acids and pigments of the fungi isolated were qualitatively determined. Total viable microbiota and bacteria concentrations were greater in February while the fungal concentration was higher in September. Aspergillus, Cladosporium, Penicillium, Curvularia and Alternaria were the predominant fungal genera in February while Cladosporium prevailed in September, although Fusarium, Mucor and Neurospora genera were also isolated in this month. The fungi isolated were capable of degrading cellulose and excreting pigments and acids. The Gram-positive bacteria group prevailed in the air and Corynebacterium, Streptomyces, Bacillus, Streptococcus, Staphylococcus, Enterobacter and Serratia were some of the genera identified.     

Production of NO2- and N2O by Nitrifying Bacteria at Reduced Concentrations of Oxygen

Pure cultures of the marine ammonium-oxidizing bacterium Nitrosomonas sp. were grown in the laboratory at oxygen partial pressures between 0.005 and 0.2 atm (0.18 to 7 mg/liter). Low oxygen conditions induced a marked decrease in the rate for production of NO₂^-, from 3.6 × 10⁻¹⁰ to 0.5 × 10⁻¹⁰ mmol of NO₂^- per cell per day. In contrast, evolution of N₂O increased from 1 × 10⁻¹² to 4.3 × 10⁻¹² mmol of N per cell per day. The yield of N₂O relative to NO₂^- increased from 0.3% to nearly 10% (moles of N in N₂O per mole of NO₂^-) as the oxygen level was reduced, although bacterial growth rates changed by less than 30%. Nitrifying bacteria from the genera Nitrosomonas, Nitrosolobus, Nitrosospira, and Nitrosococcus exhibited similar yields of N₂O at atmospheric oxygen levels. Nitrite-oxidizing bacteria (Nitrobacter sp.) and the dinoflagellate Exuviaella sp. did not produce detectable quantities of N₂O during growth. The results support the view that nitrification is an important source of N₂O in the environment.     

N2O emissions from low and moderately disturbed pasture soils—field tests of minimal and maximal N supply

Rates and patterns of nitrogen transformation differ in divergently managed pasture soils. In pastures with low nutrient inputs, N is utilized efficiently and it is assimilated by plants and soil microorganisms for synthesis of biomass. In more intensive pastures, characterized with higher N inputs, significant amounts of N can be lost from the ecosystem in various forms. Two soils of a cattle overwintering area with different levels of cattle disturbance were supplied with a solution of KNO₃ in various levels corresponding in range to 0–500 kg N ha^?1. Emissions of N₂O were measured during 24 h after a NO₃ ^?-N application. We hypothesized that under a low disturbance small additions of up to 5 kg NO₃ ^?-N are used by plants and soil microbes without an increase in N₂O emissions, while a pasture adapted to a moderate disturbance will increase N₂O emissions. Results showed that in both soils, the addition of N always increased N₂O emissions, while emissions were more pronounced in soil at the location with a higher intensity of cattle traffic. Contrary to our hypothesis, however, NO₃ ^––N was not fully metabolized in the soil with low disturbance by the cattle. Probable explanations of such a result were lower intensity of N transformations in this soil and low utilisation of N by grass. Our results suggest that under certain conditions relatively low nitrate-N inputs can also stimulate N₂O fluxes from soils.     

Method for Detection of Microorganisms That Produce Gaseous Nitrogen Oxides

A method was developed to detect NO- or N₂O-producing bacteria in solid or liquid medium by their ability to oxidize the redox indicator resazurin from its reduced colorless form to its oxidized pink form. The method was sensitive to as little as 35 nM N₂O or 0.5 nM NO. Ninety-one percent of the colonies that oxidized resazurin on plates also produced N₂O in slant cultures. Forty-four percent of the colonies that did not oxidize resazurin did produce N₂O. This percentage was reduced to 15% when colonies in which the coloration was difficult to discern were picked to slants to determine whether they oxidized the slant. The production of N₂O preceded the oxidation of resazurin by liquid cultures of Escherichia coli and a sludge isolate. With the denitrifying sewage isolate, the disappearance of N₂O was followed by the return of resazurin to its reduced state. Wolinella succinogenes was found to produce small amounts of N₂O from NO₃⁻, which resulted in a transient oxidation of resazurin.     

Soil Formate Regulates the Fungal Nitrous Oxide Emission Pathway

Fungal activity is a major driver in the global nitrogen cycle, and mounting evidence suggests that fungal denitrification activity contributes significantly to soil emissions of the greenhouse gas nitrous oxide (N₂O). The metabolic pathway and oxygen requirement for fungal denitrification are different from those for bacterial denitrification. We hypothesized that the soil N₂O emission from fungi is formate and O₂ dependent and that land use and landforms could influence the proportion of N₂O coming from fungi. Using substrate-induced respiration inhibition under anaerobic and aerobic conditions in combination with ¹⁵N gas analysis, we found that formate and hypoxia (versus anaerobiosis) were essential for the fungal reduction of ¹⁵N-labeled nitrate to ¹⁵N₂O. As much as 65% of soil-emitted N₂O was attributable to fungi; however, this was found only in soils from water-accumulating landforms. From these results, we hypothesize that plant root exudates could affect N₂O production from fungi via the proposed formate-dependent pathway.     

The potential saprotrophic capacity of foliar endophytic fungi from Quercus gambelii

Endophytic fungi occur in living tissues of terrestrial plants. Many of these fungi are primarily biotrophic, but the trophic range of endophytic fungi as a group may not be fully appreciated. In this study, our goals were (1) for the Class 3 foliar endophytic fungi isolated from Quercus gambelii, determine their potential saprotrophic capacity, which we define as the difference in growth rate in culture on Quercus gambelii leaf litter medium and control medium lacking leaf litter and (2) quantify sources of variation among isolates of these endophytic fungi in potential saprotrophic capacity, including variation due to microsite within host trees (leaves receiving full sun vs. shade) and variation within and among fungal genera. We found that 48 of the 49 tested endophytic fungal isolates have significant potential saprotrophic capacity. Contrary to expectation, the amount of solar radiation available to the leaf from which the fungi were isolated had no significant impact on potential saprotrophic capacity and there was more variability in potential saprotrophic capacity among isolates within a genus than among genera. Our results suggest that some Class 3 endophytic fungi may have the potential to function as saprotrophic fungi within plant litter, but this remains to be seen for these Quercus gambelii isolates under more natural circumstances.     

Nitrous Oxide Production by Organisms Other than Nitrifiers or Denitrifiers

Heterotrophic bacteria, yeasts, fungi, plants, and animal breath were investigated as possible sources of N₂O. Microbes found to produce N₂O from NO₃⁻ but not consume it were: (i) all of the nitrate-respiring bacteria examined, including strains of Escherichia, Serratia, Klebsiella, Enterobacter, Erwinia, and Bacillus; (ii) one of the assimilatory nitrate-reducing bacteria examined, Azotobacter vinelandii, but not Azotobacter macrocytogenes or Acinetobacter sp.; and (iii) some but not all of the assimilatory nitrate-reducing yeasts and fungi, including strains of Hansenula, Rhodotorula, Aspergillus, Alternaria, and Fusarium. The NO₃⁻-reducing obligate anaerobe Clostridium KDHS2 did not produce N₂O. Production of N₂O occurred only in stationary phase. The nitrate-respiring bacteria produced much more N₂O than the other organisms, with yields of N₂O ranging from 3 to 36% of 3.5 mM NO₃⁻. Production of N₂O was apparently not regulated by ammonium and was not restricted to aerobic or anaerobic conditions. Plants do not appear to produce N₂O, although N₂O was found to arise from some damaged plant tops, probably due to microbial growth. Concentrations of N₂O above the ambient level in the atmosphere were found in human breath and appeared to increase after a meal of high-nitrate food.     

The Earthworm Gut: an Ideal Habitat for Ingested N2O-Producing Microorganisms

The in vivo production of nitrous oxide (N₂O) by earthworms is due to their gut microbiota, and it is hypothesized that the microenvironment of the gut activates ingested N₂O-producing soil bacteria. In situ measurement of N₂O and O₂ with microsensors demonstrated that the earthworm gut is anoxic and the site of N₂O production. The gut had a pH of 6.9 and an average water content of approximately 50%. The water content within the gut decreased from the anterior end to the posterior end. In contrast, the concentration of N₂O increased from the anterior end to the mid-gut region and then decreased along the posterior part of the gut. Compared to the soil in which worms lived and fed, the gut of the earthworm was highly enriched in total carbon, organic carbon, and total nitrogen and had a C/N ratio of 7 (compared to a C/N ratio of 12 in soil). The aqueous phase of gut contents contained up to 80 mM glucose and numerous compounds that were indicative of anaerobic metabolism, including up to 9 mM formate, 8 mM acetate, 3 mM lactate, and 2 mM succinate. Compared to the soil contents, nitrite and ammonium were enriched in the gut up to 10- and 100-fold, respectively. The production of N₂O by soil was induced when the gut environment was simulated in anoxic microcosms for 24 h (the approximate time for passage of soil through the earthworm). Anoxia, high osmolarity, nitrite, and nitrate were the dominant factors that stimulated the production of N₂O. Supplemental organic carbon had a very minimal stimulatory effect on the production of N₂O, and addition of buffer or ammonium had essentially no effect on the initial N₂O production rates. However, a combination of supplements yielded rates greater than that obtained mathematically for single supplements, suggesting that the maximum rates observed were due to synergistic effects of supplements. Collectively, these results indicate that the special microenvironment of the earthworm gut is ideally suited for N₂O-producing bacteria and support the hypothesis that the in situ conditions of the earthworm gut activate ingested N₂O-producing soil bacteria during gut passage.     

Fungal diversity in ancient documents. A case study on the Archive of the University of Coimbra

This multidisciplinary research combines knowledge in molecular biology with fungal morphology, aiming at the identification of infecting fungi from historical documents on the Archive of the University of Coimbra. The identification of infecting fungi on several bibliographic documents and support materials was based both on ribosomal DNA loci amplification and sequencing, and morphological identification, using macro- and microscopical traits. A high fungal diversity was found in all types of support: parchment, laid-paper and wood-pulp paper. Fourteen fungal genera were isolated, identified, and kept in culture. The most frequent were Cladosporium, Penicillium and Aspergillus, and other less frequent genera, such as Alternaria, Botrytis, Chaetomium, Chromelosporium, Epicoccum, Phlebiopsys and Toxicocladosporium were also present. Within these genera, 20 different species were identified, from which 15 were found only in a single support type. Cladosporium cladosporioides and Penicillium chrysogenum were the only species present in all support types.     

Cultivable fungal community associated with the tropical orchid Dichaea andina

The orchid–fungus relationship has been studied since the discovery that the minute seeds of orchids depend on fungi to support the germination process. With the aim of describing the biodiversity of cultivable endophytic and mycorrhizal fungi from the orchid Dichaea andina, we isolated pure fungal cultures from its roots and identified them by sequencing the internal transcribed spacer. We recorded 22 fungal operational taxonomic units belonging to eight orders of Ascomycota: Eurotiales, Hypocreales, Xylariales, Helotiales, Boliniales, Chaetothyriales, Chaetosphaeriales and Pleosporales. The only Basidiomycota isolated belonged to the genus Ceratobasidium from the order Cantharellales, whose members are known as orchid mycorrhizal fungi. At the genus level, we identified 16 genera, the most common of which were Byssochlamys, Camarops, Trichoderma, Cladophialophora, Fusarium and Xylaria; some of them had been reported previously as orchid endophytes. The relevance of endophytic fungi to their hosts is still unclear, but this widely distributed interaction deserves further investigation.